H Fridborg

The cytotoxic activity of Taxol in primary cultures of tumour cells from patients is partly mediated by Cremophor EL.

In patient tumour samples the activity in vitro of Taxol corresponded fairly well to the known clinical activity and Taxol showed low cross-resistance to standard cytotoxic drugs. However, the Taxol solvent Cremophor EL--ethanol was considerably active alone, whereas paclitaxel formulated in ethanol was less active. Taxol thus seems to contain two components active against patient tumour cells in vitro.

Cytotoxic activity of topotecan in human tumour cell lines and primary cultures of human tumour cells from patients.

The cytotoxic activity and cross-resistance pattern of the novel topoisomerase I inhibitor topotecan (Topo) were investigated in ten cell lines, representing different mechanisms of cytotoxic drug resistance, and in 218 fresh human tumour samples using the fluorometric microculture cytotoxicity assay (FMCA). Resistance to Topo in the cell lines was associated with expression of the multidrug resistance-associated protein (MRP), whereas the cell lines with P-glycoprotein (P-gp), topoisomerase II and glutathione-associated resistance did not show decreased sensitivity to the drug. Topo was more active in haematological than in solid tumour samples, but substantial activity was observed in carcinomas of the ovary and breast, sarcoma and childhood solid tumours. Cross-resistance to standard drugs representing different mechanisms of action was generally low in patient cells. The effect of Topo was better after longer exposure, but this time-dependent effect was largely abolished when adjustment for in vitro exposure was made. Topo showed activity both in proliferative and non-proliferative cell systems. The results indicate that Topo is insensitive to major mechanisms of resistance except for MRP. Proliferation does not seem to be necessary for the effect of Topo, and no superiority for protracted dosing schedules was observed. The results also suggest that, for example, leukaemias, lymphomas, sarcomas and childhood solid tumours may be suitable targets for future phase II trials.

Activity of cyclosporins as resistance modifiers in primary cultures of human haematological and solid tumours.

The semiautomated fluorimetric microculture cytotoxicity assay (FMCA) was used for evaluation of the ability of cyclosporin A (CsA) and its novel non-immunosuppressive derivative SDZ PSC 833 (PSC) to modify the response to doxorubicin or vincristine in vitro in different haematological and solid human tumour types. Primary cultures of 322 tumour samples were analysed. Both cyclosporins showed resistance-modifying activity in all haematological tumours tested, and in solid tumours activity was observed in ovarian carcinoma and childhood tumours. Little or no effect was found in the remaining tumour types, including breast, renal and adrenal cortical carcinomas and adult sarcomas. In most of the responsive cases the interaction between the modifier and the cytotoxic drug was synergistic. There was a tendency to higher activity in samples from previously treated patients, and an inverse relationship between degree of cytotoxic drug resistance and resistance-modifying activity was noted. No difference in potency between CsA and PSC could be discerned. The results indicate differential in vitro resistance-modifying activity of the cyclosporins depending on tumour type. The results also suggest that treatment with resistance modifiers should be considered also for primary therapy of drug-sensitive tumours. Drug resistance assays such as the FMCA may become useful in preclinical evaluation of resistance modifiers.

Cytotoxic activity of calcein acetoxymethyl ester (calcein/AM) on primary cultures of human haematological and solid tumours

The aim of this study was to determine the in vitro cytotoxicity of calcein acetoxymethyl ester (Calcein/AM) on primary cultures derived from solid and haematological human tumours. Calcein/AM is a fluorescent dye that localises intracellularly after esterase-dependent cellular trapping and which has shown cytotoxic activity against various established human tumour cell lines at relatively low concentrations. The semi-automated fluorometric microculture cytotoxicity assay, based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate to fluorescein, in microtitre plates was used for the evaluation of Calcein/AM activity in tumour cell suspensions from patients. The cytotoxicity was measured as a survival index (SI), defined as the fluorescence as a percentage of control cultures. A total of 163 evaluable samples from various tumours were tested with continuous drug exposure. The activity of Calcein/AM was compared with representatives of six major classes of standard chemotherapeutic drugs. Calcein/AM was found to induce concentration-dependent decreases in the SI of both haematological and solid tumour cells. The ratio of solid over haematological tumour activity increased at a rate that was concentration dependent. Although it was relatively less active than cisplatin against solid tumours, Calcein/AM showed higher solid tumour activity compared to leukaemic specific agents (cytarabine and amsacrine), vincristine and doxorubicin (Dox). Among the solid tumours tested, childhood tumours, non-small cell lung cancer and sarcomas were the most sensitive to Calcein/AM. The best correlation between SI values was seen between Calcein/AM and Dox, with weaker correlations to representatives of antimetabolites, platinum compounds, topoisomerase II inhibitors, tubulin interactive agents and alkylators. Non-cytotoxic concentrations of cyclosporin A significantly potentiated calcein-induced cytotoxicity. The results show that Calcein/AM is differentially active against haematological tumours, but with substantial activity against solid tumours. The drug may represent a new class of anticancer compound with a unique means of drug delivery.

Differential activity of cremophor EL and paclitaxel in patients' tumor cells and human carcinoma cell lines in vitro

Previous studies indicate that Cremophor EL (CEL), the excipient for Taxol, a clinical preparation of paclitaxel, has biologic properties per se.

Relationship between pharmacokinetic parameters in patients and cytotoxicity in vitro of standard and investigational anticancer drugs.

The selection of the starting dose for initial clinical trials of anticancer agents is mostly determined by toxicological endpoints in mice (LD10). So far, very few attempts have been made to evaluate the potential value of cytotoxicity assays for this purpose. The present study was undertaken as a first attempt to Investigate the relationship between cytotoxicity of anticancer drugs in vitro and pharmacokinetic parameters in vivo in patients, at suggested maximum tolerated doses. Using the fluorometric microculture cytotoxicity assay (FMCA), we determined the concentration giving 50% cell survival (IC50) in vitro, for 25 cytotoxic drugs in fresh preparations of normal peripheral blood mononuclear cells (PBMC) and of tumor cells from patients with acute or chronic lymphocytic leukemia (ALL or CLL). Using linear regression, we investigated the relationship between the IC50s and clinically achievable peak plasma concentrations (Cmax) or concentration-time products (C × T) in humans. The clinical data was obtained from the literature. Based on all drugs tested, good correlations were obtained between IC50s for CLL cells, and both Cmax and C × T (R ≈ 0.7, p < 0.0002), and for ALL cells and normal PBMC between IC50 and Cmax, while the two latter cell types showed somewhat weaker relationships to C × T. Using the IC50 data of CLL cells, predictions of Cmax and C × T exceeded 1 log for only four drugs. No tendencies to under- or overprediction within different classes of drugs were noted. The results demonstrate a significant relationship between toxicity in vitro and achievable systemic exposure of anticancer drugs in humans, which suggests that non-clonogenic in vitro assays for drug sensitivity testing may provide pharmacokinetic information useful in the development of investigational cytotoxic drugs.

In vitro activity of 2-chlorodeoxyadenosine (CdA) in primary cultures of human haematological and solid tumours

2-Chlorodeoxyadenosine (CdA) is a deaminase-resistant purine analogue which has shown clinical activity against various haematological tumours, and is currently undergoing phase II trials. In the present study, the semiautomated fluorometric microculture cytotoxicity assay (FMCA) was used for in vitro evaluation of CdA activity in cell suspensions from both haematological and solid tumours. A total of 133 samples from various diagnoses were successfully tested with continuous drug exposure. CdA showed high in vitro activity against samples from chronic and acute lymphocytic leukaemia and acute myelocytic leukaemia, but little or no response was observed in the solid tumour groups. Cross-resistance analysis with standard drugs revealed the following rank order of correlation coefficients: cytosine arabinoside (AraC) > daunorubicin > doxorubicin > vincristine > prednisolone > 4-hydroperoxycyclophosphamide > etoposide > cisplatin. The high correlation between CdA and AraC was maintained even if the analysis was based only on the haematological tumours. The results indicate that CdA is differentially active against haematological tumours with little or no activity against solid tumours. CdA also appears highly cross resistant with AraC. If this disease-specific information is substantiated in further clinical trials and extended to other phase I-II drugs, non-clonogenic drug resistance assays such as the FMCA may become useful in new drug evaluation, and in targeting specific diagnoses and patients for phase II trials.

Cytotoxic activity of cyclosporin A and [3-keto-Bmt1]-[Val2]-cyclosporin (SDZ PSC 833) on tumour cells from patients with haematological malignancies.

AbstractObjective and method: The fluorometric microculture cytotoxic assay was employed for characterisation of the cytotoxic effect of cyclosporin A (CsA) and its non-immunosuppressive analogue SDZ PSC 833, [3-keto-Bmt1]-[Val2]-cyclosporin (PSC) in tumour cells from patients with haematological or solid tumours. Results: Tumour cells from patients with chronic lymphocytic leukaemia (CLL) or non-Hodgkins lymphoma (NHL) were found to be more sensitive to both drugs than those of tumour cells from patients with acute lymphocytic leukaemia (ALL), acute myoblastic leukaemia (AML) and various solid tumours. There was a close correlation between the effects of the two drugs (correlation coefficient 0.71), but CsA was slightly more active than PSC in most diagnoses. No tumour cell sample showed sensitivity to PSC without also being sensitive to CsA. There was a moderate level of correlation between the activity pattern of CsA and doxorubicin (correlation coefficient 0.66), whereas the correlations with other cytostatics, such as vincristine, cytarabine and melphalan, were low (correlation coefficient −0.11 to 0.33). Conclusion: The results indicate that PSC shares the direct cytotoxic properties of CsA, but is slightly less potent. Clinical testing of the cytotoxic effect of these agents in haematological malignancies seems warranted and the apparent non-cross-resistance with standard agents makes cyclosporins a potentially useful adjunct to chemotherapy in those diagnoses.

Correlation between sensitivity in vitro of patient chronic lymphocytic leukemia cells and clinical systemic exposure at the maximum tolerated dose for cell cycle phase-specific (type 2) anticancer drugs

Previously in Anti-cancer Drugs we have reported a high correlation between clinical Plasms concentration-time products (C x T) and the concentration of cytotoxic drugs giving 50% cell survival (IC50) In primary cultures of human lymphatic cells. in the present study we investigated the relationship separately for cell cycle-specific (type 1) and cell cycle non-specific (type 2) drugs in chronic lymphatic leukemla cells. A high correlation (R=0.92) was observed between C x T and IC50 for cell cycle non-specific drugs, while for cell cycle-specific, or C x T-dependent, drugs, the relationship was much weaker (R=0.58). Since the opposite pattern has been observed for the relationship between clinical C x T and LD10 in mice, these results further imply that drug sensitivity assays may be a useful complement to animal data in the selection of starting dose and dose escalation procedure in phase 1 clinical trials of new cytotoxic drugs.